Difference between revisions of "Team:British Columbia/Notebook"

Line 225: Line 225:
 
         <div id="kaempferol" class="tab-pane fade">
 
         <div id="kaempferol" class="tab-pane fade">
 
           <h3>Kaempferol Notebook</h3>
 
           <h3>Kaempferol Notebook</h3>
−
          Hello
+
       
 +
<p>
 +
We kindly received the following from UBC iGEM distribution kit of 2018:
 +
<br>
 +
 
 +
Constitutive promoter + RBS
 +
BBa_K880005
 +
~70 BP
 +
Spring 2018 Distribution
 +
Plate 2, Well 3F
 +
pSB1C3 (Cm)
 +
       
 +
<br>
 +
 
 +
F3H
 +
BBa_K1497009
 +
~1107 BP
 +
Spring 2018 Distribution
 +
Plate 2, Well 12I
 +
pSB1C3 (Cm)
 +
 
 +
<br>
 +
 
 +
 
 +
RBS
 +
BBa_B0034
 +
~12 BP
 +
Spring 2018 Distribution
 +
Plate 4, Well 1N
 +
pSB1C3 (Cm)
 +
 
 +
<br>
 +
 
 +
Double Terminator
 +
BBa_B0015
 +
~129 BP
 +
Spring 2018 Distribution
 +
Plate 3, Well 3F
 +
pSB1C3 (Cm)
 +
</p>
 +
 
 +
<br>
 +
<p>First, combining F3H and B0034:</p>
 +
<ul>
 +
<li>4CL digest with EcoRI + SpeI </li>
 +
<li>TAL digest with EcoRI + Xbal</li>
 +
<li>Ligation of both digestion with T4 ligase </li>
 +
<li> E. coli transformation via heat shock method</li>
 +
<li>Spread on CMB-LB agar plate and incubate at 37 C° overnight </li>
 +
<li>PCR and gel electrophoresis to analyze if successful </li>
 +
<li> Take positive colonies and place in LB with CMP incubate for 16 hours at 37*C </li>
 +
<li> Plasmid obtained</li>
 +
</ul>
 +
 
 +
<br>
 +
<p>Second, we will take bio bricks of CHS + CHI with RBS (ribosomal binding site): </p>
 +
<ul>
 +
<li>CHS digest with EcoRI and SpeI </li>
 +
<li> CHI digest with EcoRI and Xbal </li>
 +
<li> Ligation of both digestion with T4 ligase </li>
 +
<li> E. coli transformation via heat shock method </li>
 +
<li> Spread on CMB-LB agar plate at 37c° overnight </li>
 +
<li> PCR and gel electrophoresis to analyze if successful </li>
 +
<li> Take positive colonies and place in LB with CMP incubate for 16 hours at 37 C°</li>
 +
<li> Plasmid obtained</li>
 +
</ul>
 +
 
 +
<br>
 +
<p>Third, combine the two plasmids previously prepared:</p>
 +
<ul>
 +
<li> 4CL – TAL digest by EcoRI and SpeI</li>
 +
<li>CHS-CHI with EcoRI and Xbal </li>
 +
<li> Dephosphorylate CHS-CHI </li>
 +
<li> Ligate the two together with T4 ligation </li>
 +
<li> Add GFP via digestion and then ligation* </li>
 +
<li> E. coli transformation via heat shock method </li>
 +
<li> PCR and gel electrophoresis to analyze if successful </li>
 +
<li> Take positive colonies and place in LB and CMP incubate for 16 hours at 37 C°</li>
 +
</ul>
 +
 
 +
 
 +
<p>Protocol for cloning promoter-RBS-4CL in pSB1C3<p>
 +
A.      Restriction enzyme digest
 +
1.      Prepare the master mix in a microfuge tube by combining the following:
 +
Component
 +
Volume to add (ul)
 +
10X NEB Buffer 2.1
 +
6
 +
NEB PstI restriction enzyme
 +
4
 +
Autoclaved distilled water
 +
26
 +
TOTAL
 +
36
 +
 +
2.      Zip-spin the master mix to collect all liquid at the bottom of the tube
 +
3.      With a pipette set to 25 ul, pipette each reaction up and down several times to mix
 +
4.      Vortex the master mix thoroughly
 +
5.      Zip-spin the master mix again
 +
6.      Aliquot the master mix into 3 aliquots of 14 ul (discard the remaining master mix)
 +
7.      Label the three tubes containing the aliquots as follows:
 +
a.      P-RBS
 +
b.      4CL
 +
c.      PDC (positive digest control)
 +
 
 
         </div>
 
         </div>
  
Line 242: Line 346:
 
   });
 
   });
 
   </script>
 
   </script>
−
 
  
  

Revision as of 00:53, 18 October 2018

Naringenin Notebook

We kindly received the following from UBC iGEM distribution kit of 2018:
Constitutive promoter + RBS BBa_K880005 ~70 BP Spring 2018 Distribution Plate 2, Well 3F pSB1C3 (Cm)
BBa_K880005 ~70 BP Spring 2018 Distribution Plate 2, Well 3F pSB1C3 (Cm)
4CL BBa_K801093 ~1700 BP Spring 2018 Distribution Plate 2, Well 17D pSB1C3 (Cm)
RBS+TAL BBa_K1033000 ~1600 Spring 2018 Distribution Plate 4, Well 6C pSB1C3 (Cm)


First, TAL and 4CL with RBS (ribosomal binding site) to combine them:

  • 4CL digest with EcoRI + SpeI
  • TAL digest with EcoRI + Xbal
  • Ligation of both digestion with T4 ligase
  • E. coli transformation via heat shock method
  • Spread on CMB-LB agar plate and incubate at 37 C° overnight
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB with CMP incubate for 16 hours at 37*C
  • Plasmid obtained

Second, we will take bio bricks of CHS + CHI with RBS (ribosomal binding site):

  • CHS digest with EcoRI and SpeI
  • CHI digest with EcoRI and Xbal
  • Ligation of both digestion with T4 ligase
  • E. coli transformation via heat shock method
  • Spread on CMB-LB agar plate at 37c° overnight
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB with CMP incubate for 16 hours at 37 C°
  • Plasmid obtained

Third, combine the two plasmids previously prepared:

  • 4CL – TAL digest by EcoRI and SpeI
  • CHS-CHI with EcoRI and Xbal
  • Dephosphorylate CHS-CHI
  • Ligate the two together with T4 ligation
  • Add GFP via digestion and then ligation*
  • E. coli transformation via heat shock method
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB and CMP incubate for 16 hours at 37 C°

Protocol for cloning promoter-RBS-4CL in pSB1C3

A. Restriction enzyme digest 1. Prepare the master mix in a microfuge tube by combining the following: Component Volume to add (ul) 10X NEB Buffer 2.1 6 NEB PstI restriction enzyme 4 Autoclaved distilled water 26 TOTAL 36 2. Zip-spin the master mix to collect all liquid at the bottom of the tube 3. With a pipette set to 25 ul, pipette each reaction up and down several times to mix 4. Vortex the master mix thoroughly 5. Zip-spin the master mix again 6. Aliquot the master mix into 3 aliquots of 14 ul (discard the remaining master mix) 7. Label the three tubes containing the aliquots as follows: a. P-RBS b. 4CL c. PDC (positive digest control)

Biosensor Notebook

Kaempferol Notebook

We kindly received the following from UBC iGEM distribution kit of 2018:
Constitutive promoter + RBS BBa_K880005 ~70 BP Spring 2018 Distribution Plate 2, Well 3F pSB1C3 (Cm)
F3H BBa_K1497009 ~1107 BP Spring 2018 Distribution Plate 2, Well 12I pSB1C3 (Cm)
RBS BBa_B0034 ~12 BP Spring 2018 Distribution Plate 4, Well 1N pSB1C3 (Cm)
Double Terminator BBa_B0015 ~129 BP Spring 2018 Distribution Plate 3, Well 3F pSB1C3 (Cm)


First, combining F3H and B0034:

  • 4CL digest with EcoRI + SpeI
  • TAL digest with EcoRI + Xbal
  • Ligation of both digestion with T4 ligase
  • E. coli transformation via heat shock method
  • Spread on CMB-LB agar plate and incubate at 37 C° overnight
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB with CMP incubate for 16 hours at 37*C
  • Plasmid obtained

Second, we will take bio bricks of CHS + CHI with RBS (ribosomal binding site):

  • CHS digest with EcoRI and SpeI
  • CHI digest with EcoRI and Xbal
  • Ligation of both digestion with T4 ligase
  • E. coli transformation via heat shock method
  • Spread on CMB-LB agar plate at 37c° overnight
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB with CMP incubate for 16 hours at 37 C°
  • Plasmid obtained

Third, combine the two plasmids previously prepared:

  • 4CL – TAL digest by EcoRI and SpeI
  • CHS-CHI with EcoRI and Xbal
  • Dephosphorylate CHS-CHI
  • Ligate the two together with T4 ligation
  • Add GFP via digestion and then ligation*
  • E. coli transformation via heat shock method
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB and CMP incubate for 16 hours at 37 C°

Protocol for cloning promoter-RBS-4CL in pSB1C3

A. Restriction enzyme digest 1. Prepare the master mix in a microfuge tube by combining the following: Component Volume to add (ul) 10X NEB Buffer 2.1 6 NEB PstI restriction enzyme 4 Autoclaved distilled water 26 TOTAL 36 2. Zip-spin the master mix to collect all liquid at the bottom of the tube 3. With a pipette set to 25 ul, pipette each reaction up and down several times to mix 4. Vortex the master mix thoroughly 5. Zip-spin the master mix again 6. Aliquot the master mix into 3 aliquots of 14 ul (discard the remaining master mix) 7. Label the three tubes containing the aliquots as follows: a. P-RBS b. 4CL c. PDC (positive digest control)

Plasmid Maintenance Notebook